The use of stem cells in the form of a cell-based therapies is currently one of the most exciting and promising areas for disease treatment and reparative medicine. Clearly, basic research into the ways by which proliferation and differentiation of e.g. embryonic and adult stem cells can be controlled is vitally important.
U.S. Pat. No. 6,548,264 describes silica coated nanoparticles which comprise a magnetic metal core. The magnetic core present in the particles enables the particles to be responsive to a magnetic field and therefore, the particles are suitable for use in diagnostic, imaging and recording systems. However, the nanoparticles of the prior art may suffer from the disadvantage that they do not define the method of activation at a cellular level.
Magnetic bead twisting cytometry has been used to define the mechanical properties of single cells and to demonstrate that external mechanical forces can be transmitted across the cell surface and through the cytoskeleton via transmembrane cell adhesion molecules such as integrins, see, for example, Wang, N and Ingberger, D E (1995) Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry. Biochem. Cell Biol. 73: 327-335.
There have been many developments in biocompatible magnet nanoparticle synthesis, characterization1-3 and applications of novel magnetic techniques in the field of healthcare4-6. This work primarily has involved investigating the controlled and directed transport of pharmaceuticals. In these systems therapeutic drugs or genes may be attached to magnetic carrier particles (usually polymer coated magnetite), which are then concentrated at the target site in vivo by the application of spatially focused, high gradient magnetic fields. Once the drug/carrier complexes have accumulated at the target site, the drug is released and uptake at the sites is enhanced. Investigations have been made into new methods for magnetic targeting for gene therapy as well as theoretically and experimentally examining and improving deposition of magnetic micro- and nanoparticle carriers in model systems in vitro and in vivo4,6.
Short-term experiments where force is applied to the cell membrane using torque or where tension is applied to transmembrane proteins such as RGD or collagen molecules has been described by a number of researchers7,8. These experiments use ‘mechanical’ stimulation of the membrane to trigger short term internal calcium fluxes in a variety of cells. It is known that mechanical signalling using other techniques can trigger differentiation pathways in bone marrow stromal cells down the osteogenic lineage11 and in particular, that low level mechanical signals across the membrane can up-regulate expression and DNA binding activity of osteoblastic specific transcription factors, cbfal and cfos12,13.
In these investigations, force can be applied to a number of different tagged receptors. It has been demonstrated how we can influence downstream processes and enhance collagen and other matrix protein synthesis15. Using bone marrow derived mesenchymal stem cells conditioned to differentiate along the osteogenic and chondrogenic lineage we have been investigating downstream gene regulation in response to magnetic particle activation of specific receptors. Preliminary data has shown an up-regulation in Runx 2 in response to magnetic particle stimulation of calcium channels in human mesenchymal stem cells followed by up-regulation of a mechanosensitive matrix protein, osteopontin. In addition, we have evidence of up-regulation of SOX 9 following stimulation of monolayer human dedifferentiated chondrocytes. These studies have been extended to 3D analysis of cell-seeded scaffolds over long-term culture to investigate the use of these strategies for construct fabrication in tissue engineering in vitro. Furthermore, preliminary studies which include a dose-response analysis of particle number and force applied are encouraging and indicate increased matrix synthesis and expression of the osteogenic phenotype14.
Bone marrow contains multipotential stromal stem cells or mesenchymal stem cells which can differentiate into, inter alia, fibroblastic, osteogenic, adipogenic and reticular cells. These mesenchymal stem cells, such as human bone marrow stromal fibroblasts can be isolated from volunteer donors and may retain their multilineage (adipocytic, chondrogenic, osteoblastic) potential. One advantage in the use and manipulation of the aforementioned cells lies in their lack of immunogenicity which provides the potential for use of these cells in, inter alia, cartilage and bone repair.
Our as yet unpublished co-pending International Patent Application, No. PCT/GB2003/002624 combines the magnetic nanoparticle approach with knowledge of mechanosensitive ion channels, in particular, the TREK K+ channel. It is established that the TREK channel is present in osteogenic, chondrogenic and bone marrow stromal cells In order to define more closely the targeting of specific receptors to control activation, we have used HIS-tagged clones of the TREK gene. HIS tags have been inserted into particular regions of the TREK molecule to allow attachment of HIS antibody or NI2+ bound magnetic particles which can then be remotely torques using a magnetic field. Sites of the ion channel protein which lie both internal and external to the cell membrane have been tagged and in this way we can identify the mechanosensitive regions of the molecule as well as define the signal frequencies required to switch on downstream processes. FIG. 2 shows the results of experiments using bone marrow stromal cells with internal calcium levels up-regulated as a result of the application of magnetic fields to magnetic nanoparticles attached to a His-tagged TREK channel.
It has been shown that conditioning connective tissue cells in vitro can be achieved, by, inter alia, the development of a magnetic force bioreactor which enables magnetic fields to be applied in vitro to 2D monolayer cultures and 3D cell-seeded scaffolds.
However, neither US '264 nor Wang solve or even address the problems surrounding two fundamental questions which need to be addressed, and which encompass the ultimate goal of engineering cells for clinical use, namely;    (i) how will cells be targeted to the site of repair and held at that site; and    (ii) how will cells e.g. stem cells, be conditioned or differentiated in vitro and/or in vivo.